When less is more: Combining site-specific isotope labeling and NMR unravels structural details of huntingtin repeats.

In this issue of Structure, Urbanek et al. (2020a) combine site-specific isotope labeling and NMR spectroscopy to investigate opposing effects of flanking regions onto the conformation of the poly-Q region in Huntingtin. Poly-Q interactions with preceding residues promote an a-helical conformation while a following proline-rich region favors extended conformations

Combining NMR, SAXS and SANS to characterize the structure and dynamics of protein complexes.

Understanding the structure and dynamics of biological macromolecules is essential to decipher the molecular mechanisms that underlie cellular functions. The description of structure and conformational dynamics often requires the integration of complementary techniques. In this review, we highlight the utility of combining nuclear magnetic resonance (NMR) spectroscopy with small angle scattering (SAS) to characterize these challenging biomolecular systems. NMR can assess the structure and conformational dynamics of multidomain proteins, RNAs and biomolecular complexes. It can efficiently provide information on interaction surfaces, long-distance restraints and relative domain orientations at residue-level resolution. Such information can be readily combined with high-resolution structural data available on subcomponents of biomolecular assemblies. Moreover, NMR is a powerful tool to characterize the dynamics of biomolecules on a wide range of timescales, from nanoseconds to seconds. On the other hand, SAS approaches provide global information on the size and shape of biomolecules and on the ensemble of all conformations present in solution. Therefore, NMR and SAS provide complementary data that are uniquely suited to investigate dynamic biomolecular assemblies. Here, we briefly review the type of data that can be obtained by both techniques and describe different approaches that can be used to combine them to characterize biomolecular assemblies. We then provide guidelines on which experiments are best suited depending on the type of system studied, ranging from fully rigid complexes, dynamic structures that interconvert between defined conformations and systems with very high structural heterogeneity

Client binding shifts the populations of dynamic Hsp90 conformations through an allosteric network.

Hsp90 is a molecular chaperone that interacts with a specific set of client proteins and assists their folding. The underlying molecular mechanisms, involving dynamic transitions between open and closed conformations, are still enigmatic. Combining nuclear magnetic resonance, small-angle x-ray scattering, and biochemical experiments, we have identified a key intermediate state of Hsp90 induced by adenosine triphosphate (ATP) binding, in which rotation of the Hsp90 N-terminal domain (NTD) yields a domain arrangement poised for closing. This ATP-stabilized NTD rotation is allosterically communicated across the full Hsp90 dimer, affecting distant client sites. By analyzing the interactions of four distinct clients, i.e., steroid hormone receptors (glucocorticoid receptor and mineralocorticoid receptor), p53, and Tau, we show that client-specific interactions with Hsp90 select and enhance the NTD-rotated state and promote closing of the full-length Hsp90 dimer. The p23 co-chaperone shifts the population of Hsp90 toward the closed state, thereby enhancing client interaction and processing

Selectivity, cocrystal structures, and neuroprotective properties of leucettines, a family of protein kinase inhibitors derived from the marine sponge alkaloid leucettamine B.

DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) are implicated in the onset and development of Alzheimer's disease and Down syndrome. The marine sponge alkaloid leucettamine B was recently identified as an inhibitor of DYRKs/CLKs. Synthesis of analogues (leucettines) led to an optimized product, leucettine L41. Leucettines were cocrystallized with DYRK1A, DYRK2, CLK3, PIM1, and GSK-3β. The selectivity of L41 was studied by activity and interaction assays of recombinant kinases and affinity chromatography and competition affinity assays. These approaches revealed unexpected potential secondary targets such as CK2, SLK, and the lipid kinase PIKfyve/Vac14/Fig4. L41 displayed neuroprotective effects on glutamate-induced HT22 cell death. L41 also reduced amyloid precursor protein-induced cell death in cultured rat brain slices. The unusual multitarget selectivity of leucettines may account for their neuroprotective effects. This family of kinase inhibitors deserves further optimization as potential therapeutics against neurodegenerative diseases such as Alzheimer's disease

NudC guides client transfer between the Hsp40/70 and Hsp90 chaperone systems.

In the eukaryotic cytosol, the Hsp70 and the Hsp90 chaperone machines work in tandem with the maturation of a diverse array of client proteins. The transfer of nonnative clients between these systems is essential to the chaperoning process, but how it is regulated is still not clear. We discovered that NudC is an essential transfer factor with an unprecedented mode of action: NudC interacts with Hsp40 in Hsp40-Hsp70-client complexes and displaces Hsp70. Then, the interaction of NudC with Hsp90 allows the direct transfer of Hsp40-bound clients to Hsp90 for further processing. Consistent with this mechanism, NudC increases client activation in vitro as well as in cells and is essential for cellular viability. Together, our results show the complexity of the cooperation between the major chaperone machineries in the eukaryotic cytosol